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Copyright © 2007 The Royal Society Interaction between host genotype and environmental conditions affects bacterial density in Wolbachia symbiosis *Author and address for correspondence: Zoologisches Institut der Universität Basel-Evolutionsbiologie, Vesalgasse 1, 4051 Basel, Switzerland (Email: laurence.mouton/at/unibas.ch) Received December 1, 2006; Accepted December 14, 2006. | |||||||||
Abstract Regulation of microbial population density is a necessity in stable symbiotic interactions. In Wolbachia symbiosis, both bacterial and host genotypes are involved in density regulation, but environmental factors may also affect bacterial population density. Here, we studied the interaction between three strains of Wolbachia in two divergent homozygous lines of the wasp Leptopilina heterotoma at two different temperatures. Wolbachia density varied between the two host genotypes at only one temperature. Moreover, at this temperature, reciprocal-cross F1 insects displayed identical Wolbachia densities, which were intermediate between the densities in the two parental lines. While these findings confirm that the host genotype plays an important role in Wolbachia density, they also highlight its interaction with environmental conditions, making possible the evolution of local adaptations for the regulation of Wolbachia density. | |||||||||
1. Introduction Symbiotic population density is a key factor of host–symbiont relationships, because it influences both the efficiency of transmission and the virulence of the symbiont. When symbionts are vertically transmitted, host and symbionts may act on the regulation of symbiont density to optimize the trade-off between these two parameters (Ebert & Bull 2003). While theoretical works are numerous in this field, empirical studies are by far insufficient, especially in systems where symbionts are vertically transmitted. Recently, Wolbachia bacteria have become a recognized model system for the study of symbiotic regulation. Wolbachia are widespread bacterial endosymbionts of arthropods and filarial nematodes that display various types of interaction with their hosts, ranging from mutualism to reproductive parasitism, including cytoplasmic incompatibility (CI). Mechanisms that control Wolbachia density involve interactions between hosts and bacteria (Kondo et al. 2005). Indeed, within a given multiply infected host, the density of each strain can differ, demonstrating that the Wolbachia genotype plays a role in the regulation process (Ijichi et al. 2002; Ikeda et al. 2003; Mouton et al. 2003, 2004). The involvement of the host genotype on the regulation of Wolbachia density has also been demonstrated, both at an interspecific level by transinfection experiments (Ikeda et al. 2003) and at an intraspecific level by cytological analysis in Drosophila species (Clark et al. 2003) and quantitative PCR in the adzuki bean beetle Callosobruchus chinensis (Kondo et al. 2005). In addition to the genetic influence of both partners, environmental factors can also affect symbiont density. Among these factors, temperature is especially important in ectotherms, and its influence on host–symbiont interactions in general, and in Wolbachia symbiosis in particular, has been documented previously (reviewed in Thomas & Blanford 2003; Mouton et al. 2006). The combination of genetic and environmental factors can lead to complex genotype-by-genotype-by-environment interactions as already reported in other symbiotic systems (Greub et al. 2003), but data are lacking in Wolbachia symbiosis. Here, we study the density of Wolbachia in two homozygous lines of the parasitic wasp Leptopilina heterotoma (Hymenoptera: Figitidae) at two different temperatures. These lines display a high level of genetic divergence (Fleury et al. 1995), but naturally harbour the same three CI-Wolbachia strains (Vavre et al. 1999). This study confirms the role of the host genotype on the bacterial population, but also demonstrates that environmental conditions may locally affect the evolution of the association, due to the existence of host genotype-by-environment interactions on the control of bacterial populations. | |||||||||
(a) Insect strains and rearing Leptopilina heterotoma, a solitary endoparasitoid wasp of Drosophila species, developed in a Wolbachia-free strain of Drosophila melanogaster originating from Lyon (France) on standard diet (David 1962) at 20 or 26°C with an LD cycle of 12 :12 and 70% relative humidity.Two inbred lines were used: A7, originated from Antibes (France, 43.5° (b) Quantification DNA was extracted from the whole body of 5-day-old individuals using Chelex solution (Biorad) as in Mouton et al. (2003).Wolbachia cell number was measured using the quantitative PCR LightCycler System (Roche Diagnostics). The Wolbachia surface protein gene wsp was amplified using either generalist primers or the specific primers of each Wolbachia strain as described in Mouton et al. (2003). Wolbachia load was calculated by correcting the numbers of Wolbachia cells by the fresh weight of each insect. This allows size variations of the wasps associated with their genetic background and their rearing temperature to be taken into account. (c) Experiment 1: influence of temperature on host genotype–Wolbachia strain interaction We studied the influence of host genotype on Wolbachia regulation within two environments by measuring the total and specific Wolbachia densities in female wasps of both strains reared either at 20 or 26°C (five individuals measured for each condition). Both lines had been kept at these temperatures for at least two generations before the experiments were performed.(d) Experiment 2: Wolbachia density in reciprocal F1 hybrids For each cross (parental lines and reciprocal F1), 1–2 day old virgin females were mated with 4–5 day old males (15 repetitions per condition). After mating (visually checked), three females were each provided with 100 Drosophila larvae and allowed to parasitize them for 24h. After developing at 20°C, the wasps that emerged were individually tested to determine their Wolbachia load as above. | |||||||||
3. Results (a) Experiment 1: influence of temperature on host genotype–Wolbachia strain interaction The Wolbachia load was determined in A7 and SF4 females at 20 and 26°C (table 1). At 20°C, SF4 females harbour more Wolbachia than A7 females, but in contrast there is no difference between the two genotypes at 26°C. This demonstrates a clear interaction between the host genotype and the environment (ANOVA2: F1,16=6.41, p=0.022).
We also measured the specific load of the three Wolbachia strains (figure 1). Globally, the effect on each strain is comparable to the effect observed on total load with a reduced load only in A7 females reared at 20°C. Consequently, the proportion of each strain remains constant whatever the conditions (table 2). wLhet3 accounted for the highest fraction of the total Wolbachia load, and wLhet2 the lowest, which is consistent with data obtained previously at 20°C on A7 individuals (Mouton et al. 2003).
(b) Experiment 2: Wolbachia density in reciprocal F1 hybrids at 20°C As in the first experiment, SF4 females harbour more Wolbachia than A7 females (Student's t-test: p=0.0015). The Wolbachia load of the two reciprocal F1 hybrid females are equivalent (p=0.616) and intermediate between the values found for the A7 and SF4 parent females (figure 2).
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4. Discussion (a) Influence of the host genotype on Wolbachia density At 20°C, SF4 females harbour more Wolbachia than A7 females, and reciprocal F1 hybrid females show the same level of infection, intermediate between the two parental lines. Thus, variation between the two insect lines must be attributable to intrinsic differences in the host genotype, as previously demonstrated by Kondo et al. (2005). The host may control the population of bacteria either directly, by limiting the nutrients provided to them or by actively eliminating bacterial cells, or indirectly through the response of the bacterial population to its own physiological state. Whatever the mechanism involved, it is clear that host genetic characteristics do influence Wolbachia density.(b) Influence of temperature on host genotype–Wolbachia strain interaction The higher Wolbachia density in SF4 than in A7 females is observed at 20°C but not at 26°C. This difference is due to the lack of any response of the SF4 females to temperature, since they harbour the same Wolbachia load at 20 and 26°C. On the other hand, the effect of the A7 genotype on Wolbachia differs between these two temperatures, suggesting a different ability to influence bacterial density according to temperature. Moreover, despite the fact that the relative proportions of the three strains do not vary with temperature conditions, they do not contribute equally to the total Wolbachia density since each strain has a specific density, indicating the role of their genotype on this basal level. These observations indicate that Wolbachia load depends on the Wolbachia genotype, the host genotype and the environment.(c) How can we account for within-species differences in Wolbachia? Two recent studies have demonstrated a positive correlation between Wolbachia density and the cost of infection (McGraw et al. 2002; Mouton et al. 2004). Moreover, temperature has been shown to play a major role in Wolbachia density and, as a consequence, in its transmission (Hurst et al. 2001). Influence of bacterial density on the phenotypic expression of the association, together with the influence of host genetic background on bacterial load in a given environment, provides the necessary conditions for the evolution of local adaptations. In the present case, the average annual temperatures of the original locations of insect lines are 20°C (Antibes, A7) and 17°C (Lyon, SF4), respectively (data: Meteo France). Global physiological adaptation of the insects to local climatic differences could account for their differing capacities to regulate bacterial density in response to temperature change. It is of course premature to speculate about field situations, and further experiments involving more than two populations of L. heterotoma and a wider range of temperatures are needed.Bacterial density is of great importance in the evolution of symbiotic associations, and this study highlights that interactions between hosts and Wolbachia strongly depend on the host genotype, but also on the temperature conditions, as had been claimed previously (Weeks et al. 2002). Therefore, host genotype-by-environment interactions could play a major role in the expression of the phenotype, and thus on the evolution of host–Wolbachia associations. | |||||||||
Acknowledgments This study was partly supported by Centre National de la Recherche Scientifique (IFR 41-UMR5558). We thank the DTAMB for technical assistance. Isabelle Colson improved the language. | |||||||||
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